A New Herbal Combination, Etana, for Enhancing Erectile Function: an Efficacy and Safety Study in Animals

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ORIGINAL ARTICLE A new herbal combination, Etana, for enhancing erectile function: an efficacy and safety study in animals

N Qinna1,3,HTaha2, KZ Matalka1 and AA Badwan3

1Department of Pharmacology and Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, Petra University, Amman, Jordan; 2Delass Natural Products, Naor, Jordan and 3Central Research Lab, The Jordanian Pharmaceutical Manufacturing Co. PLC (JPM), Naor, Jordan

We present herein a new herbal combination called Etana that is composed of five herbal extracts including Panax quinquelotius (Ginseng), Eurycoma longifolia (Tongkat Ali),Epimedium grandiflorum (Horny goat weed), Centella asiatica (Gotu Kola) and flower pollen extracts. Most of the above-mentioned extracts have a long historical and traditional use for erectile dysfunction (ED). On the basis of the mechanism of action of each of the above, a combination is introduced to overcome several physiological or induced factors of ED. This study was conducted to show an enhancement of erectile function in male rats. The animals were observed for 3 h after each administration for penile erection, genital grooming and copulation mounting, and the penile erection index (PEI) was calculated. The maximum response was observed at the concentration of 7.5 mg kg–1 of Etana. At a 7.5 mg kg–1 single dose, the percentage of responding rats was 53±7witha PEI of 337±72 compared with 17±6 with a PEI of 30±10 for control animals. This PEI was significantly (Po0.001) higher than each single component and than the sum of any two herbal components of Etana. When compared with sildenafil citrate, Etana induced more pronounced PEI than 0.36 mg kg–1, but similar to 0.71 mg kg–1 of sildenafil. Furthermore, full acute and sub-acute toxicity studies showed no toxic effects of Etana. In conclusion, this study describes a new and safe combination of herbal components that enhance erectile function in male rats. Clinical studies are warranted for evaluating Etana’s significance in ED. International Journal of Impotence Research (2009) 21, 315–320; doi:10.1038/ijir.2009.18; published online 4 June 2009

Keywords: Ginseng; Centella; Epimedium; Eurycoma; pollen; erectile dysfunction; Etana; herbal

Introduction ED transiently, such as the orally consumed phos- phodiesterase type 5 inhibitors, testosterone ther- Erectile dysfunction (ED) affects 50% of men aged apy, or vasoactive agents inserted intraurethrally or between 40 and 70 years and therefore is considered injected intracavernosally.1–3 to be an important health problem.1 As men age, Some natural products such as Panax quinquelo- several physiological or induced factors arise that tius, Eurycoma longifolia and Epimedium grand- contribute to ED, such as a decline in the testicular iflorum have the ability to act as an aphrodisiac and production of testosterone, vascular functionality, to help restore ED. The medicinal activity of Panax levels and responsiveness to vasoactive amines and quinquelotius (Ginseng) has improved penile neurotransmitters, diseases (for example, cardio- rigidity, libido and patient satisfaction in men with vascular, hypertension, diabetes mellitus, chronic ED,4,5 whereas using a Eurycoma longifolia extract prostatitis) and certain drugs.1,2 A number of (Tongkat Ali) and Epimedium grandiflorum (Horny pharmacological agents are introduced to correct Goat Weed) in animals increased sexual arousal, motivation and frequency of sexual activity.6–10 Furthermore, there are some natural products that could play a role in improving circulation to the Correspondence: Professor AA Badwan, Central Research prostate and penis such as Centella asiatica (Gotu Lab, The Jordanian Pharmaceutical Manufacturing Co. 11–13 PLC (JPM), P.O. Box 94, Naor, Naor 11710, Jordan. Kola) and flower pollen. Therefore, it was our E-mail: [email protected] hypothesis that the development of a herbal combi- Received 28 January 2009; revised 22 April 2009; accepted nation of the above five plant extracts, called 22 April 2009; published online 4 June 2009 Etana,14 could work on several age-induced causes Efficacy and safety study in animals N Qinna et al 316 of ED. On the basis of the mechanism of action of Sexual behavior and penile erection index each component, this herbal combination could Each animal group consisted of 10 male rats have an additive or synergistic effect to restore weighing 200–300 g. Each test preparation erectile function. was dissolved freshly in distilled water and doses To introduce Etana as an enhancer of male erectile were administered by oral gavage. Dosing of function, this study examines the efficacy of Etana Etana was either as a single dose per day or as a in relation to each of its components, to its triple dose per day, 3 h apart, to show any changes dose–response effect, in comparison to sildenafil in the efficacy obtained from multiple administra- as a known drug to restore erectile function and tions per day. Control animals were given the to different herbal combinations. In addition, acute vehicle alone (distilled water). Rats were placed in and subacute toxicity studies of Etana were glass cages, allowed free access to food and carried out to establish the safety of this herbal tap water and were observed for 3 h after each combination. drug administration for penile erection, genital grooming and possible copulation mounting. The number of responding rats was recorded along Materials and methods with the number of sexual activity episodes (penile erection, genital grooming or copulation mounting). Herbal extracts Penile erection index (PEI) was calculated for each Panax quinquelotius, Eurycoma longifolia and group by multiplying the percentage of active rats (responding rats) by the total number of activity Epimedium grandiflorum extracts were purchased 15–17 from Hongjiu Ginseng, the Active Ingredients episodes. Group and from the Chengdu Wagott Pharmaceuti- cal Co., Chengdu, China, respectively. Centella asiatica (Gotu Kola) and flower pollen extracts were Acute and subacute toxicity assessment purchased from Graminex, USA and Ennagram, Acute toxicity for Etana was determined in rats France, respectively. Sildenafil citrate was obtained (250–300 g) consisting of 10 rats per group (five from JPM, Jordan. males and five females). A single dose of 0, 7.5, 37.5, 75, 150, 225 and 300 mg kg–1 (that is, 1 Â to 40 Â of the human recommended daily dose based on 70 kg b.w.) was given by oral gavage to each animal per Etana preparation and method of analysis group. The animals were observed closely for any Etana is a mixture of Ginseng extract (100 mg), toxic or abnormal behavior in the first 2 h after Tongkat Ali extract (200 mg), Epimedium extract dosing and were kept under further observation for 2 (50 mg), Gotu Kola extract (40 mg), and flower weeks. pollen extract (135 mg). The preparation was A subacute toxicity study for 28 days was carried dissolved in distilled water and given to rats by oral out according to ICH guidelines. A single dose of 0, gavage. 7.5, 15 and 75 mg kg–1 (that is, 1 Â ,2Â ,10Â of the The method of analysis of Etana components is human recommended daily dose based on 70 kg based on the HPLC method to assay a marker for b.w.) was given by oral gavage to each animal per each constituent (for example, icariin for Epime- group. Each test group consisted of five males and dium grandiflorum and malasiatic acid for Centella five females, and different sex animals were kept in asiatica). The method is based on a solid stationary separate cages to avoid pregnancy during the test phase (C18 packed column), mobile phase, and period. Animals were monitored carefully and body separation by partition adsorption or ion exchange weights were measured daily. At the end of 28 days, process. The gradient mixtures of acetonitrile:H3PO4 all animals were killed. Just before being killed, and the detection wavelengths were different for blood samples were taken from the jugular vein for a each component. full blood and chemistry analysis. All internal organs were carefully removed, weighed and then fixed with 10% buffered formalin for histological Animals examination. Male and female Wister rats (220–300 g) were obtained from the Yarmouk University animal house unit (Irbid, Jordan). The animals were housed at the Data analysis animal facility in Petra University in a 12 h light or All variables were analyzed using SPSS version 10 dark cycle at a constant temperature of 22 1C. All statistical package (SPSS Inc., Chicago, IL, USA) animals were acclimatized for 10 days before the using different statistical tests. For sexual behavior experiments with free access to a standard diet and and PEI analysis, Student’s t test was carried out to drinking water. All animal experiments were compare the level of significance between groups. carried out in compliance with relevant laws and As for the toxicity study, statistics were generated institutional guidelines. for time interaction, gender effect and differences

International Journal of Impotence Research Efficacy and safety study in animals N Qinna et al 317 500 1500 400 300 1000 200 Average PEI

100 Daily PEI 500 0 Control Ginseng Tongkat Ali Gotu kola Epimedium F. Pollen Etana 0 Figure 1 The PEI for different groups of rats administered 0 5 10 15 20 either a single component of Etana, Etana or distilled water. The PEI was determined by multiplying the average scores reported Dose of Etana (mg/kg) during a 6-day treatment by the average percentage of responding Figure 2 The PEI for different groups of rats administered rats. The rats were monitored for 3 h after a single administration. different doses of Etana. Etana was administered thrice a day, 3 h Each single group was administered the same amount (mg) as is apart, and PEI was determined by multiplying the average scores present in Etana. Each bar point represents the mean of six ± reported during a 3-day treatment by the average percentage of experiments s.d. responding rats. Each bar point represents the mean of three experiments±s.d. between each treatment group and the control group. The overall differences between the groups were analyzed using one-way ANOVA. In some 2500 cases, Tukey’s post test was carried out after ANOVA to show the differences between selected groups. 2000 For all of the statistical comparisons, the level of significant difference was defined as Po0.05. 1500

1000 Daily PEI

Results 500

Etana versus each single component on male rat 0 sexual behavior Control Sildenafil Sildenafil Etana The PEI after the administration of each single 0.36 mg/kg 0.71mg/kg 7.5 mg/kg component of Etana in comparison with Etana is Figure 3 The PEI for different groups of rats administered two presented in Figure 1. In all treated animals, PEI doses of sildenafil and Etana. Doses were administered thrice a increased significantly (Po0.001) when compared day, 3 h apart, and PEI was determined by multiplying the average scores reported during a 3-day treatment by the average with control animals. In addition, Etana-treated rats percentage of responding rats. Each bar point represents the showed significantly higher (Po 0.001) PEI than did mean of three experiments±s.d. each single Etana component-treated rats (Figure 1). In addition, the PEI of Etana is higher than the sum of any two herbal components. Furthermore, the therapeutic doses (0.36 and 0.71 mg kg–1, based on number of responding rats after a single administra- 70 Kg b.w.) of sildenafil citrate. The PEI after Etana tion of Etana was significantly higher (Po0.001) –1 (7.5 mg kg ) administration as a single or triple dose than each single Etana component-treated rat. per day was similar to that of 0.71 mg kg–1 of sildenafil and was significantly higher (Po0.001) than that of 0.36 mg kg–1 of sildenafil and the control Dose response of Etana on male rat sexual behavior group (Figure 3). The dose response of Etana showed a significantly higher (Po0.001) PEI at a dose of 7.5 mg kg–1 of –1 Etana when compared with 2.5, 15 mg kg Etana versus a different mixtures of herbal and controls (Figure 2). In addition, when Etana components on male rat sexual behavior was administered thrice a day, 3 h apart to the same The PEI after administration of different mixture rats, the PEI was significantly higher (Po0.001) at –1 components, group 1 (Centella asiatica, Eurycoma 7.5 mg kg dose when compared with that in the longifolia, Epimedium grandiflorum, flower pollen other doses and control, and the cumulative PEI did extract and Gingko) and group 2 (Ginseng, Euryco- not change after the second or third dose to the same ma longifolia and Epimedium grandiflorum), is rats (Figure 2). shown in Figure 4. The cumulative PEI and the percentage of responding rats after the administration of Etana thrice a day were significantly higher Etana versus Sildenafil on rat sexual behavior (Po0.001) than the PEI and percentage of respond- In this set of experiments, the effect of Etana ing rats of group1 and 2 (Figure 4). In group 1, 7.5 mg kg–1 was compared with the effect of two Gingko was added instead of Ginseng because of its

International Journal of Impotence Research Efficacy and safety study in animals N Qinna et al 318 known function as an aphrodisiac. A single admin- (Table 1). The reduction of cholesterol was dose istration of Gingko (0.86 mg kg–1) showed PEI and dependent (Po0.001) as the percent reduction was percentage of responding rats at 80 and 22%, 20, 26 and 34% for 7.5, 15 and 75 mg kg–1, respectively. respectively, whereas the reduction of urea was dose independent (15% for all doses; Po0.03) and the reduction of potassium was seen only at the 75 mg kg–1 dose (10%; Po0.015). On the other hand, Acute and subacute toxicity of Etana the glucose level increased significantly only at No deaths occurred after the administration of any 75 mg kg–1 dose (68%, Po0.025) (Table 1). of the single doses tested (7.5–300 mg kg–1). After a –1 The hematological changes after a 28-day admin- 28-day administration of 7.5, 15 or 75 mg kg (that istration of 7.5, 15 and 75 mg kg–1 dose of Etana is, 1 Â ,2Â ,10Â of the effective dose), no deaths showed a significant increase in the percentage occurred, and the body weight did not show any of lymphocytes, and a significant decrease in the significant changes in male or female rats. In percentage of neutrophils in peripheral blood at addition, the weights of the internal organs did not doses of 15 and 75 mg kg–1 (Po0.05–0.001) (Table 1). show any changes after a 28-day administration of However, the absolute number of the above cells in any of the doses tested. peripheral blood did not change because there was The chemistry results after a 28-day administra- an apparent reduction in the total leukocytes count. tion of 1 Â ,2Â , and 10 Â dose of Etana showed no significant differences in triglycerides, ALT, AST, ALP, sodium, creatinine, calcium and phosphorus. However, a significant reduction in cholesterol, urea Discussion and potassium levels (Po0.03–0.001) was observed This study describes a new and safe combination of herbal components that enhances erectile function 2500 in male rats. Most of the single constituents of Etana have been widely used for enhancing erectile function, and scientific evidence was reported to 2000 explain the mechanism of each component. The idea was to show the additive or synergistic effect of 1500 such combination. The results indicate that Etana showed a significantly higher percentage of re- 1000 sponding rats and PEI. Furthermore, the Etana Daily PEI efficacy was dose dependent, showing higher activity at either single dose or triple dose of 500 7.5 mg kg–1 per day, and can be administered for a long period of time without any toxic effect. 0 To confirm our hypothesis with regard to the Control Group 1* Group 2* Etana efficacy of Etana combination versus other possibi- lities, it was compared with two other combinations. Figure 4 The PEI for different groups of rats administered The choice of the two other combinations was based different combinations: Centella asiatica, Eurycoma longifolia, Epimedium grandiflorum, flower pollen extract and Gingko for on the known mechanism of each component. group 1: and Ginseng, Eurycoma longifolia and Epimedium Group1 was a mixture of Centella asiatica, Euryco- grandiflorum for group 2 and Etana. Doses were administered ma longifolia, Epimedium grandiflorum, pollen thrice a day, 3 h apart, and PEI was determined by multiplying the extract and Gingko versus Ginseng, Eurycoma long- average scores reported during a 3-day treatment by the average percentage of responding rats. Each single group was adminis- ifolia and Epimedium grandiflorum (group 2) and tered the same amount (mg) as is present in Etana. Each bar point Etana (Ginseng, Eurycoma longifolia, Epimedium represents the mean of three experiments±s.d. grandiflorum, Centella asiatica and flower pollen).

Table 1 The significant hematological and biochemical findings of rats treated with different doses of Etana for 28 days

Group Cholesterol Urea Potassium Glucose Lymphocytes % Neutrophils % (mg dl–1) (mg dl–1) (mmol l–1) (mg dl–1)

Control 58±237±2 6.5±0.2 55±973±313±2 7.5 mg kg–1 46±5* 31±2* 6.7±0.2 56±570±314±2 15 mg kg–1 41±3** 32±2* 6.8±0.3 75±380±2* 8±1* 75 mg kg–1 38±2** 33±1* 5.8±0.2* 92±12* 87±2* 5±1*

*Po0.05 when compared with the control group, **Po0.001 when compared with the control group.

International Journal of Impotence Research Efficacy and safety study in animals N Qinna et al 319 Group 1 components are similar to Etana except that after a long day of use. In addition, this herbal it contains Gingko instead of Ginseng. Gingko has combination could also help in reducing the serum also been used for aphrodisiac effects but it has a cholesterol level and in managing chronic inflam- different mechanism of action from Ginseng.18 mation of the prostate.11–13 Clinical studies are Group 2 does not contain flower pollen and Centella warranted for evaluating Etana’s significance in ED asiatica. On the basis of the above, one of the and in men with chronic prostatitis. mechanisms of action of Etana as a herbal combination to enhance blood flow is consistent with the synergistic effects observed by combining the in- Conflict of Interest dividual components.11–13 On the basis of the published scientific evidence The authors declare no conflict of interest. of each Etana component, the mechanism of action of Etana can be fourfold. First, it has been shown that ginsenosides, which are extracted from Panax Acknowledgments ginseng, increased the plasma levels of FSH, LH, testosterone (total and free forms) and spermatozoa This work was funded by The Jordanian Pharma- concentration and motility.4 This suggests that ceutical Manufacturing Co. PLC (JPM), Naor, Jordan. ginsenosides act on the hypothalamus and or pituitary to increase plasma FSH and LH, thus activating testes to increase testosterone levels and spermatozoa formation.4,19 Second, it was found References that Eurycoma longifolia enhanced the testosterone 1 McVary KT. Erectile dysfunction. New Eng J Med. 2007; 357: effect by increasing the sexual performance of 2472–2481. 6 inexperienced castrated male rats. Third, it has 2 Rosen RC, Link CL, O’Leary MP, Giuliano F, Aiyer LP, been shown that ginsenosides and icariin, isolated Mollon P. Lower urinary tract symptoms and sexual health: from Epimedium grandiflorum, promoted the re- the role of gender, lifestyle and medical comorbidities. BJU Int 2009; 103: Suppl 3: 42–47. lease of nitric oxide (NO) from corpus caverno- 10,20 3 Feifer A, Carrier S. Pharmacotherapy for erectile dysfunction. sum. The release of NO induces the relaxation of Expert Opin Investig Drugs 2008; 17: 679–690. the smooth muscle and thus enhances erection. In 4 Salvati G, Genovesi G, Marcellini L, Paolini P, De Nuccio I, addition, ginsenosides and icariin were found to Pape M et al. Effects of Panax Ginseng C. A Meyer saponins on 10,20 male fertility. Panminerva Med 1996; 38: 249–254. increase intracavernosal pressure. Furthermore, 5 de Andrade E, de Mesquita AA, Claro Jde A, de Andrade PM, icariin was found to be a cGMP-specific phos- Ortiz V, Paranhos M et al. Study of the efficacy of Korean Red 24 phodiesterase 5 inhibitor in vitro, but not in vivo, Ginseng in the treatment of erectile dysfunction. Asian after oral dosing for 4 weeks.10 In this study, J Andol 2007; 9: 241–244. however, the dose response of Etana showed a 6 Ang HH, Cheang HS, Yusof AP. Effects of Eurycoma longifolia Jack (Tongkat Ali) on the initiation of sexual performance in bell-shaped curve (Figure 2), suggesting a phospho- inexperienced castrated male rats. Exp Anim 2000; 49: 35–38. diesterase inhibition. Fourth, the addition of flower 7 Ang HH, Lee KL. Effect of Eurycoma longifolia Jack on pollen extract and Centella asiatica improves blood orientation activities in middle aged male rats. Fundam Clin circulation to the prostate and penis, thereby Pharmacol 2002; 16: 479–483. enhancing the level of the other components (or 8 Ang HH, Lee KL, Kiyoshi M. Eurycoma longifolia Jack 11–13 enhances sexual motivation in middle – aged male mice. their effects) of Etana to reach the genital tract. J Basic Clin Physol Pharmacol 2003; 14: 301–308. Furthermore, it is known that one of the major 9 Gu Y, Meng G. Preparation conditions for decoction of problems that could result in ED is chronic Epimedium grandiflorum Morr. Zhongguo Zhong Yao Za Zhi prostatitis.2,13 Both Centella asiatica (Gotu Kola) 1990; 15: 412–413, 446. 10 Liu WJ, Xin ZC, Xin H, Yaun YM, Tian L, Guo YL. Effects of and flower pollen have antioxidative activities that icariin on erectile dysfunction and expression of nitric oxide are important to reduce male infertility and help in synthase isoforms in castrated rats. Asian J Andol 2005; 7: managing chronic prostatitis.11,13 381–388. In addition to enhancing erectile function, Etana 11 Brinkhaus B, Linder M, Schuppan D, Hahn EG. Chemical, pharmacological and clinical profile of the East Asian medical has other benefits. It lowered the serum cholesterol plant Centella asiatica. Phytomedicine 2000; 7: 427–428. level after 28 days of oral dosing in a dose- 12 Pointel JP, Boccalon H, Cloarec M, Ledevehat C, Joubert M. dependent manner. This cholesterol-lowering effect Titrated extract of Centella asiatica (TECA) in the treatment of is mainly due to Panax ginseng and flower pollen.21– venous insufficiency of the lower limbs. Angelology 1986; 37: 23 It has been shown that Panax ginseng lowers 420–421. 13 Chen HJ, Wang ZP, Chen YR, Qin DS, Fu SJ, Ma BL. Effects cholesterol and triglyceride levels by activating 22 of pollen extract EA-10, P5 on chronic prostatitis or infertility lipoprotein lipase in hyperlipidemic rats. In this with chronic prostatitis. Acta Pharmacol Sin 2002; 23: study, however, the rats were normal and 28 days of 1035–1039. Etana administration did not cause any significant 14 Badwan A, Taha H, Qinna N. Multi-component herbal composition for the treatment of male erectile dysfunction. change in the triglyceride levels. Patent Pending Appl #. EP20070001673, Publ:08/06/2008; This paper describes a new combination of herbal International Classes A61K36/258; A61K36/185; A61K36/296; extracts that enhances erectile function and is safe A61K36/25; A61P15/10.

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